Identiﬁcation and Characterization of Fusarium Species Causing Watermelon Fruit Rot in Northern Thailand

: Fruit rot caused by phytopathogenic fungi is one of the major diseases affecting water-melons ( Citrullus lanatus ) around the world, which can result in unmarketable fruits and signiﬁcant economic losses. Fruit rot was observed on watermelons throughout the postharvest storage periods in Phayao Province, northern Thailand in 2022. For the present study, a total of ten fungal isolates were isolated from the rot lesions of watermelons. All obtained fungal isolates were then characterized in terms of their pathogenicity. The results indicated that only four fungal isolates caused rot disease with similar symptoms during the postharvest storage period. Based on their morphological characteristics, these four fungal isolates were identiﬁed as belonging to the genus Fusarium . Using multi-gene phylogenetic analyses with a combination of the translation elongation factor 1-alpha ( tef-1 ), calmodulin ( cam ), and RNA polymerase second largest subunit ( rpb2 ) genes, the fungal isolates were subsequently identiﬁed as Fusarium compactum and F. paranaense . Taken together, the results of this study indicate that F. compactum and F. paranaense cause fruit rot disease in watermelons. To the best of our knowledge, this is the ﬁrst study to report F. compactum and F. paranaense as novel pathogens of watermelon fruit rot both in Thailand and elsewhere in the world.


Introduction
Watermelon (Citrullus lanatus) is one of the most significant economic fruits within the family Cucurbitaceae. It has successfully been planted and farmed in subtropical and tropical regions throughout the world [1][2][3]. In 2022, the Food and Agriculture Organization Statistical Database (FAOSTAT) [4] demonstrated that China was the world's largest producer of watermelons producing 60.25 million tonnes (with global production recorded at 101.62 million tonnes), followed by Turkey, India, Iran, and Algeria. This increasing trend in watermelon production is expected to continue into the future. In Southeast Asia, watermelon production in the area is led by Vietnam followed by Indonesia, the Lao People's Democratic Republic, Thailand, and the Philippines [4]. Many scientific studies have reported that watermelon fruits are a good source of nutrition for humans. They contain a variety of important nutrients, including amino acids, carbohydrates, fiber, minerals, organic acids, proteins, sugars, and vitamins [5][6][7]. Furthermore, watermelon fruits contain several beneficial chemical compounds, including alkaloids, flavonoids, glycosides, phenols, tannins, terpenoids, saponins, and steroids, which possess advantageous pharmacological properties [8,9]. These compounds can be utilized in therapeutic approaches due to their antimicrobial, anticancer, antiulcer, antioxidant, anti-inflammatory, antihypertensive, analgesic, and antigiardial properties, which allow them to function against prosthetic to moderate (22-40% of disease infection on fruit areas) infection by rot symptoms. The lesions on the watermelon fruit gradually expand and combine to encompass the whole fruit, providing the infected fruit with a bruised, decayed, and broken appearance. The internal area of decomposition seemed obviously degraded and the surrounding tissues were soaked with water (Figure 1d,e).

Fungal Isolation
Ten fungal isolates (FPY1 to FPY10) were isolated from the watermelons that were collected and which exhibited the typical rot symptoms. Subsequently, all fungal isolates were inoculated into asymptomatic commercial watermelons. The conidia collected from two-week-old cultures on potato dextrose agar (PDA) of each fungal isolate were used as the inoculum. A conidial suspension of each fungal isolate was individually dropped onto the wounded fruits at the equator of each fruit. After three days of conidial inoculation, only four fungal isolates-namely, FPY1, FPY4, FPY7, and FPY9-led to the development of rot lesions. The disease symptoms of these four fungal isolates are provided below. All four fungal causal agents-namely, FPY1, FPY7, FPY4, and FPY9-were stored in 20% glycerol and submitted to the culture collection of the Sustainable Development of Biological Resources (SDBR-CMU), Faculty of Science, Chiang Mai University, Thailand, with the numbers SDBR-CMU461, SDBR-CMU462, SDBR-CMU463, and SDBR-CMU464, respectively. These four fungal isolates were selected for further species identification.

Fungal Isolation
Ten fungal isolates (FPY1 to FPY10) were isolated from the watermelons that were collected and which exhibited the typical rot symptoms. Subsequently, all fungal isolates were inoculated into asymptomatic commercial watermelons. The conidia collected from two-week-old cultures on potato dextrose agar (PDA) of each fungal isolate were used as the inoculum. A conidial suspension of each fungal isolate was individually dropped onto the wounded fruits at the equator of each fruit. After three days of conidial inoculation, only four fungal isolates-namely, FPY1, FPY4, FPY7, and FPY9-led to the development of rot lesions. The disease symptoms of these four fungal isolates are provided below. All four fungal causal agents-namely, FPY1, FPY7, FPY4, and FPY9-were stored in 20% glycerol and submitted to the culture collection of the Sustainable Development of Biological Resources (SDBR-CMU), Faculty of Science, Chiang Mai University, Thailand, with the numbers SDBR-CMU461, SDBR-CMU462, SDBR-CMU463, and SDBR-CMU464, respectively. These four fungal isolates were selected for further species identification.

Morphological Observations
Four fungal isolates (SDBR-CMU461, SDBR-CMU462, SDBR-CMU463, and SDBR-CMU464) were selected and used in this experiment. Fungal colonies of each isolate were observed on oatmeal agar (OA), PDA, and synthetic nutrient-poor agar (SNA) at 25 • C for one week. According to the fungal colony characteristics, the isolate SDBR-CMU461 was related to the isolate SDBR-CMU462, whereas the isolate SDBR-CMU463 was related to the isolate SDBR-CMU464. All fungal isolates produced both macro-and micro-conidia, as well as chlamydospores. Based on these morphological features, all the isolated fungi were initially determined to be members of the genus Fusarium [35][36][37][38]. The identification of the fungi was subsequently confirmed by multi-gene phylogenetic analyses.

Phylogenetic Analysis
The sequences derived from the four fungal isolates obtained in this investigation were submitted to the GenBank database (Tables 1 and 2). Based on the BLAST results, two fungal isolates-namely, SDBR-CMU461 and SDBR-CMU462-belonged to the F. incarnatum-equiseti species complex, whereas the fungal isolates SDBR-CMU463 and SDBR-CMU464 belonged to the F. solani species complex. Fungal identification was further confirmed through subsequent multi-gene phylogenetic analyses. Two phylogenetic trees (for F. incarnatum-equiseti and F. solani species complexes) were constructed in this study. The results of both phylogenetic analyses revealed that the topological results of both the maximum likelihood (ML) and Bayesian inference (BI) analyses employed in each analysis were similar (data not shown). Consequently, the phylogenetic trees generated by the ML analysis are presented. Ex-type, epi-type, and neotype species are indicated by the superscript letters as "T", "ET," and "NT," respectively. The symbol "−" indicates the absence of sequencing information in GenBank.  Ex-type, epi-type, and neotype species are indicated by the superscript letters as "T", "ET", and "NT", respectively. The symbol "−" indicates the absence of sequencing information in GenBank.
For phylogenetic analysis of the F. incarnatum-equiseti species complex, the combined tef-1, cam, and rpb2 sequence data set was used, according to the identification techniques used in previous studies [35][36][37][38]. The aligned data set contained 2181 bp including gaps (tef-1: 1-704, cam: 705-1288, and 127 rpb2: 1289-2181) with 45 taxa. The outgroup consisted of F. camptoceras and F. neosemitectum from the F. camptoceras species complex (FCAMSC). A phylogenetic tree is represented in Figure 2. Our phylogenetic tree was constructed with the aim of having similar outcomes to previous phylogenetic studies [13,[35][36][37][38]. The phylogenetic tree assigned the two fungal isolates (SDBR-CMU461 and SDBR-CMU462) assessed in this investigation within the same clade of F. compactum, which consisted of the type species CBS 186.31 in the F. equiseti clade. This clade established a monophyletic clade with high statistical support (100% BS and 1.0 PP). Fusarium compactum formed a species that was phylogenetically related to F. lacertarum. Therefore, these two fungal isolates (SDBR-CMU461 and SDBR-CMU462) were identified as F. compactum. The combined tef-1 and rpb2 sequence data set was used for phylogenetic analy the F. solani species complex, following the identification techniques employed in e studies [50,55]. This phylogenetic analysis included 41 taxa and the aligned data se tained 1415 bp including gaps (tef-1: 1-603 and rpb2: 604-1415). The outgroup consis F. decemcellulare and F. setosum from the F. decemcellulare species complex (FDSC). A logenetic tree of the F. solani species complex is shown in Figure 3. Our phylogenet was constructed with the aim of being similar to those in previous phylogenetic s [50,55,56]. The phylogenetic tree successfully assigned the two fungal isolates (S CMU463 and SDBR-CMU464) assessed in this investigation within the same clade paranaense, which consisted of the type species CML 1830. This clade established a m phyletic clade with high statistical support (99% BS and 1.0 PP). Fusarium para formed a sister taxon to F. falciforme with high statistical support (97% BS and 1. The combined tef-1 and rpb2 sequence data set was used for phylogenetic analysis of the F. solani species complex, following the identification techniques employed in earlier studies [50,55]. This phylogenetic analysis included 41 taxa and the aligned data set contained 1415 bp including gaps (tef-1: 1-603 and rpb2: 604-1415). The outgroup consisted of F. decemcellulare and F. setosum from the F. decemcellulare species complex (FDSC). A phylogenetic tree of the F. solani species complex is shown in Figure 3. Our phylogenetic tree was constructed with the aim of being similar to those in previous phylogenetic studies [50,55,56]. The phylogenetic tree successfully assigned the two fungal isolates (SDBR-CMU463 and SDBR-CMU464) assessed in this investigation within the same clade of F. paranaense, which consisted of the type species CML 1830. This clade established a monophyletic clade with high statistical support (99% BS and 1.0 PP). Fusarium paranaense formed a sister taxon to F. falciforme with high statistical support (97% BS and 1.0 PP). Thus, both fungal isolates (SDBR-CMU463 and SDBR-CMU464) were recognized as F. paranaense. Colonies on PDA were yello white in the center, white at the margins, and flat with undulated edges that were yellow. Colonies on OA were greyish yellow in the center, white at the margins, had aerial mycelia, and were flat with entire edges that were greyish orange. Colonies on were white and umbilicated with entire edges that were white. Pigment and odor not present. Sporodochia were not found in any agar media. Conidiophores were fo white in the center, white at the margins, and flat with undulated edges that were pale yellow. Colonies on OA were greyish yellow in the center, white at the margins, had dense aerial mycelia, and were flat with entire edges that were greyish orange. Colonies on SNA were white and umbilicated with entire edges that were white. Pigment and odor were not present. Sporodochia were not found in any agar media. Conidiophores were formed on aerial mycelium, of a size of 12.5-100 × 2.8-4.2 µm, which appeared as branched, and bore terminal or lateral phialides. Phialides were monophialidic, subulate to sub-cylindrical, hyaline, smooth and thin-walled, and of a size of 13.1-31.6 × 2.6-4.3 µm. Chlamydospores were abundant, globose, ellipsoid, intercalarily or terminal, hyaline to pale yellow with age, smooth-walled, solitary, in chains or clusters, and of a size of 6.6-17.4 × 6.1-16.7 µm (av. ± SD: 11.1 ± 2.5 × 11.0 ± 2.4 µm). Microconidia were abundant, hyaline, oval to ellipsoidal, straight to slightly curved, aseptate, and of a size of 5.3-13.5 × 2.1-3.7 µm (av. ± SD: 9.6 ± 1.9 × 2.8 ± 0.3 µm). Macroconidia hyaline were thick-walled, strongly curved, had 1-7-septa, and were of a size of 13.3-72.5 × 3.3-6.4 µm (av. ± SD: 33.0 ± 13.1 × 4.6 ± 0.6 µm).  Note: Morphologically, the two isolates of F. compactum obtained in this study could produce microconidia, which has not been recorded in previous studies [57,58]. However, their other morphological characteristics agreed well with the previous descriptions of F. compactum [57,58]. Phylogenetically, F. compactum forms a species that is phylogenetically related to F. lacertarum. However, F. lacertarum may be distinguished from F. compactum by its shorter conidiophores (up to 7.0 µm long) and phialides (2.5-4.0 × 1.0-1.5 µm) [59]. Note: Morphologically, the two isolates of F. compactum obtained in this study could produce microconidia, which has not been recorded in previous studies [57,58]. However, their other morphological characteristics agreed well with the previous descriptions of F. compactum [57,58]. Phylogenetically, F. compactum forms a species that is phylogenetically related to F. lacertarum. However, F. lacertarum may be distinguished from F. compactum by its shorter conidiophores (up to 7.0 µm long) and phialides (2.5-4.0 × 1.0-1.5 µm) [59]. Colonies on OA, PDA, and SNA grew to 80-83, 75.0-78.0, and 77.0-80.5 mm in diameter, respectively, at 25 • C in the dark for one week. Colonies on PDA were orangewhite in the center, white at the margins, and flat with entire edges that were light yellow. Colonies on OA were brownish orange in the center and white at the margins with aerial mycelia that were dense and flat with entire edges that were brownish orange. Colonies on SNA were white and raised with entire edges that were white. Pigment and odor were not present. Sporodochia were not found in any agar media. Conidiophores were formed on aerial mycelium, of a size of 12-105 × 2.5-4.1 µm, were verticillately branched, and bore terminal or lateral phialides. Phialides were monophialidic, subulate to subcylindrical, hyaline, smooth and thin-walled, and of a size of 10.8-38.9 × 2.3-5.4 µm. Chlamydospores were abundant, hyaline, globose, intercalarily or terminal, ellipsoid, smooth to rough-walled, solitary, or were present in pairs or formed chains, and of a size of 6.2-11.3 × 6.2-11.6 µm (av. ± SD: 9.2 ± 1.4 × 8.9 ± 1.3 µm). Microconidia were abundant, hyaline, thin-walled, elongated to ellipsoidal, straight to slightly curved, aseptate, and of a size of 5.3-20.1 × 2.3-5.2 µm (av. ± SD: 11.4 ± 3.4 × 4.0 ± 0.7 µm). Macroconidia were hyaline, cylindrical to fusiform, 1-4-septate, and of a size of 16.0-40.6 × 3.5-5.4 µm (av. ± SD: 29.1 ± 6.5 × 4.7 ± 0.4 µm). Colonies on OA, PDA, and SNA grew to 80-83, 75.0-78.0, and 77.0-80.5 mm in diameter, respectively, at 25 °C in the dark for one week. Colonies on PDA were orangewhite in the center, white at the margins, and flat with entire edges that were light yellow. Colonies on OA were brownish orange in the center and white at the margins with aerial mycelia that were dense and flat with entire edges that were brownish orange. Colonies Note: The morphological characteristics of isolates SDBR-CMU463 and SDBR-CMU464 corresponded to descriptions of F. paranaense [50]. Phylogenetically, F. paranaense forms a sister taxon to F. falciforme; however, the growth of F. paranaense appeared to be slower than that of F. falciforme (85.0 mm) on PDA for one week at 25 • C [18]. In addition, F. paranaense produces elongated to ellipsoidal microconidia, whereas F. falciforme produces oval microconidia [18].

Pathogenicity Test
The disease symptoms of F. compactum (SDBR-CMU461 and SDBR-CMU462) and F. paranaense (SDBR-CMU463 and SDBR-CMU464) are shown in Figures 6 and 7, respectively. Primary symptoms appeared on the wounded fruits as small light-brown to brown spots and developed into green bruises. After that, these spots developed into dark green bruised spots that were covered with a dense white mycelia for F. compactum (Figure 6b,c) and a thin white mycelia for F. paranaense (Figure 7b,c) surrounding each lesion. The inoculated fruits displayed moderate infections, as characterized by rot symptoms after one week of incubation. A cross section of a mature lesion indicated that the interior lesion area seemed to be decomposing and was encircled by water-soaked tissue (Figures 6e,f and 7e,f). Following a 14-day inoculation period, the lesions covered the entire fruit. The fruits eventually became extremely rotten and squashy. The symptoms of the disease were consistent with those observed during the postharvest storage period. Nevertheless, no disease symptoms were observed on wounded fruits treated with sterile distilled water (Figures 6a,d and 7a,d). Each fungal isolate was consistently re-isolated from all inoculated tissues and re-identified using both morphological methods of characterization in order to fulfill Koch's postulates. F. paranaense produces elongated to ellipsoidal microconidia, whereas F. falciforme produces oval microconidia [18].

Pathogenicity Test
The disease symptoms of F. compactum (SDBR-CMU461 and SDBR-CMU462) and F. paranaense (SDBR-CMU463 and SDBR-CMU464) are shown in Figures 6 and 7, respectively. Primary symptoms appeared on the wounded fruits as small light-brown to brown spots and developed into green bruises. After that, these spots developed into dark green bruised spots that were covered with a dense white mycelia for F. compactum (Figure 6b,c) and a thin white mycelia for F. paranaense (Figure 7b,c) surrounding each lesion. The inoculated fruits displayed moderate infections, as characterized by rot symptoms after one week of incubation. A cross section of a mature lesion indicated that the interior lesion area seemed to be decomposing and was encircled by water-soaked tissue (Figures 6e,f and 7e,f). Following a 14-day inoculation period, the lesions covered the entire fruit. The fruits eventually became extremely rotten and squashy. The symptoms of the disease were consistent with those observed during the postharvest storage period. Nevertheless, no disease symptoms were observed on wounded fruits treated with sterile distilled water (Figures 6a,d and 7a,d). Each fungal isolate was consistently re-isolated from all inoculated tissues and re-identified using both morphological methods of characterization in order to fulfill Koch's postulates.

Discussion
Fusarium is considered to be one of the most important genera of plant pathogens, as it is known to cause serious diseases in several economic plants-including watermelons-grown around the world [16,60,61]. Traditional approaches to the characterization and identification of the Fusarium species are mainly based on morphological characteristics [38,58,62]. Due to the wide variety of morphological differences, it is impossible to distinguish between the closely related Fusarium species based on morphological characteristics alone [38,58]. Therefore, molecular methods are essential to concretely identify Fusarium at the species level. An effective method for identifying Fusarium species has been designed using protein-coding (β-tubulin, cam, tef-1, and RNA polymerase largest sub-unit) and ribosomal DNA (the internal transcribed spacer and the large sub-unit regions) genes [35,38,42,[63][64][65][66]. However, several previous studies have reported that species-level identification of Fusarium cannot be achieved using only the ribosomal DNA gene [67,68]. Therefore, the accurate identification of Fusarium species is currently carried out using a combination of morphological characteristic and multi-gene molecular phylogenetic analyses [35][36][37][38]40,63,64,66]. In this study, two isolates of F. compactum (SDBR-CMU461 and SDBR-CMU462) and two isolates of F. paranaense (SDBR-CMU463 and SDBR-CMU464) were isolated from fruit rot lesions on watermelons from northern Thailand. These four fungal isolates were identified using a combination of their morphological features and phylogenetic analysis of multiple genes, according to the identification techniques used in previous studies [35][36][37][38]50,55,56]. Prior to this study, F. compactum and F. paranaense had previously been identified as plant pathogens; for example, F. compactum was found to be the cause of leaf spot on sweet cherry (Prunus avium L.) [69] and leaf blight on maize (Zea mays L.) [70] in China, root rot of banana (Musa sp.) in Greece [71], and canker of Italian cypress (Cupressus sempervirens) trees in Israel [72]. In Brazil, F. paranaense caused root rot in soybeans [Glycine max (L.) Merr.] [50].
The pathogenicity of all F. compactum and F. paranaense isolates in this study was examined in order to confirm Koch's postulates. According to the results, both fungal spe-

Discussion
Fusarium is considered to be one of the most important genera of plant pathogens, as it is known to cause serious diseases in several economic plants-including watermelonsgrown around the world [16,60,61]. Traditional approaches to the characterization and identification of the Fusarium species are mainly based on morphological characteristics [38,58,62]. Due to the wide variety of morphological differences, it is impossible to distinguish between the closely related Fusarium species based on morphological characteristics alone [38,58]. Therefore, molecular methods are essential to concretely identify Fusarium at the species level. An effective method for identifying Fusarium species has been designed using protein-coding (β-tubulin, cam, tef-1, and RNA polymerase largest subunit) and ribosomal DNA (the internal transcribed spacer and the large sub-unit regions) genes [35,38,42,[63][64][65][66]. However, several previous studies have reported that species-level identification of Fusarium cannot be achieved using only the ribosomal DNA gene [67,68]. Therefore, the accurate identification of Fusarium species is currently carried out using a combination of morphological characteristic and multi-gene molecular phylogenetic analyses [35][36][37][38]40,63,64,66]. In this study, two isolates of F. compactum (SDBR-CMU461 and SDBR-CMU462) and two isolates of F. paranaense (SDBR-CMU463 and SDBR-CMU464) were isolated from fruit rot lesions on watermelons from northern Thailand. These four fungal isolates were identified using a combination of their morphological features and phylogenetic analysis of multiple genes, according to the identification techniques used in previous studies [35][36][37][38]50,55,56]. Prior to this study, F. compactum and F. paranaense had previously been identified as plant pathogens; for example, F. compactum was found to be the cause of leaf spot on sweet cherry (Prunus avium L.) [69] and leaf blight on maize (Zea mays L.) [70] in China, root rot of banana (Musa sp.) in Greece [71], and canker of Italian cypress (Cupressus sempervirens) trees in Israel [72]. In Brazil, F. paranaense caused root rot in soybeans [Glycine max (L.) Merr.] [50].
The pathogenicity of all F. compactum and F. paranaense isolates in this study was examined in order to confirm Koch's postulates. According to the results, both fungal species can be regarded as causal agents of fruit rot disease in watermelons. Our results are supported by previous studies that reported that Fusarium species are the cause of various disease symptoms in watermelons in tropical and subtropical regions around the world [73][74][75][76]. Prior to this study, F. solani, F. oxysporum, F. verticillioides, and F. chlamydosporum were considered to be the causal agents of fruit rot in watermelons in Nigeria [16,24,77]. In particular, F. equiseti was found to cause fruit rot in watermelons cultivated in China [78], Malaysia [26], and the United States [79]. Postharvest fruit rot found on watermelons that was caused by F. falciforme and F. oxysporum has also been reported in Malaysia [18]. Furthermore, other Fusarium species have also been associated with the severity of several watermelon diseases. For example, F. equiseti and F. oxysporum f. sp. niveum have been observed to cause Fusarium wilt disease in fruits grown in Korea [76] and Malaysia [74], respectively. On the other hand, Fusarium brachygibbosum and F. oxysporum have been shown to lead to vine decline symptoms in the United States [73] and root rot in China [75], respectively. Furthermore, other fungal species from the genera Alternaria, Aspergillus, Curvularia, Fusarium, Macrophomina, Phytophthora, Lasiodiplodia, Sclerotium, and Pythium have also been associated with fruit rot in watermelons. For example, Pythium aphanidermatum and P. debaryanum caused fruit rot disease in watermelons collected in China [20]; Phytophthora capsici was found to cause fruit rot in watermelons in China [20] and the United States [22]; Kwon and Park [21] found that Sclerotium rolfsii caused postharvest fruit rot in watermelons in South Korea and, in Nigeria, Alternaria cucumeria, Aspergillus flavus, Curvularia lunata, Lasiodiolodia theobromae, and Macrophomina phaseolina have been identified as causal agents of postharvest fruit rot in watermelon [23,24].
In Thailand, the Fusarium species has been associated with symptoms of fruit rot in a number of fruits. For example, fruit rot in cantaloupes and muskmelons has been associated with F. equiseti [34], F. incarnatum [80], and F. melonis [13]. Fusarium fabicercianum caused fruit rot disease in mangoes (Mangifera indica Linn.) [81]. Cases of fruit rot in lychee (Litchi chinensis Sonn) [82] and durian (Durio zibethinus Murray) fruits [83] have been found to be caused by F. solani. Prior to this study, only incidences of watermelon fruit rot caused by F. citrullicola have been reported in Thailand [13]. The symptoms of fruit rot disease caused by F. compactum and F. paranaense in watermelons are similar to those determined to have been caused by known fungal pathogens [13,21,22,25]; however, to date, there have been no reports of watermelon fruit rot caused by F. compactum and F. paranaense. Therefore, we propose that F. compactum and F. paranaense should be identified as new pathogens of watermelon fruit rot in Thailand and throughout the world. Follow-up study is required to clarify the source of the disease inoculum and how weather conditions influence infection and disease development with respect to these pathogens. Furthermore, determination of the incidence of this disease in other areas of Thailand and throughout the world is a necessary task.

Sample Collection
Ten watermelon fruits (Citrullus lanatus) with typical rot symptoms were collected during the postharvest storage periods in Phayao Province, northern Thailand (19 • 08 20 N, 99 • 54 42 E) in 2022 (two periods: February to May and mid-October to December). All symptomatic fruits were randomly selected and placed in sterile plastic boxes. After being transported to the laboratory, the symptomatic fruits were described and assessed under a stereomicroscope (Nikon H55OS, Tokyo, Japan).

Fungal Isolation
All symptomatic fruits were processed to isolate the fungal causal agents by storing them in a plastic container with moistened filter paper to stimulate fungal conidia production. The single conidial isolation technique was used to isolate the causal fungi from rot lesions on 1.0% water agar supplemented with streptomycin (0.5 mg/L) under a stereomicroscope, following the methods established by Choi et al. [84]. After 24-48 h of incubation at 25 • C in the dark, individual germ conidia were selected and transferred directly onto PDA (CONDA, Madrid, Spain) including streptomycin (0.5 mg/L). Pure fungal isolates were kept in 20% glycerol and submitted to the culture collection of the SDBR-CMU, Chiang Mai Province, Thailand.

Pathogenicity Tests
Conidia collected from two-week-old cultures on PDA of each fungal isolate were used in this experiment. Asymptomatic commercial watermelons were thoroughly washed and their surfaces were disinfected by immersion in sterile 1.5% (v/v) NaOCl solution for 5 min. Subsequently, sterile distilled water was used to rinse them three times. After being surface-disinfected, the fruits were air-dried for 10 min at room temperature (25 ± 2 • C) [85]. The equator of each fruit received a uniform wound (5 pores, 1 mm width and 1 cm depth) with an aseptic needle after being air-dried [13]. A conidial suspension (500 µL, 1 × 10 6 conidia/mL) of each fungal isolate was separately dropped onto the wounded fruits. Subsequently, the wounded fruits were inoculated with a drop of sterile distilled water as a control. The inoculated fruit was then kept under conditions of 80% relative humidity in a separate sterile plastic container (26 × 35.5 × 20 cm). The plastic containers were kept in a growth chamber at 25 • C during a 12 h light phase for a week. All treatments were repeated twice with ten replicates of each treatment. The samples were assessed according to the degree of disease infection on the damaged fruit areas, with scores ranging from 1-25% (mild), 26-50% (moderate), 51-75% (severe), to 76-100% (extremely severe) [86]. To confirm Koch's postulates, the fungi were again isolated from any lesions that appeared on the inoculated fruits using the single spore isolation technique described above. The single spore isolation technique previously mentioned was employed to re-isolate the fungi from any lesions that appeared on the inoculated fruits in order to confirm Koch's postulates.

Morphological Studies
Colony characteristics of the fungal isolates on OA (Difco, Le Pont de Claix, France), PDA, and SNA were observed following incubation in darkness at 25 • C for a week, according to the methods described in previous studies [35,36,38]. Micromorphological features were assessed and photographed using a light microscope (Nikon Eclipse Ni-U, Tokyo, Japan). The size information related to the anatomical properties (e.g., chlamydospores, conidiogenous cells, conidiophores, phialides and conidia) were measured with at least 50 numbers of each structure using the Tarosoft (R) Image Frame Work program.

DNA Extraction, Amplification, and Sequencing
The genomic DNA of each week-old fungal isolate cultivated on PDA at 25 • C was extracted using a DNA extraction kit (FAVORGEN, Ping-Tung, Taiwan). Polymerase chain reaction (PCR) was employed to amplify the tef-1, cam, and rpb2 genes using the primer pairs EF1/EF2 [87], CAL-228F/CAL-2Rd [88], and RPB2-5F2/RPB2-7cR [65], respectively. The three genes' amplification programs were carried out in independent PCR reactions, consisting of an initial denaturation for 3 min at 95 • C, followed by 35 cycles of denaturation for 30 s at 95 • C, annealing steps for 50 s at 60 • C (tef-1), 30 s at 59 • C (cam) or 1 min at 52 • C (rpb2), and a final extension step for 1 min at 72 • C on a peqSTAR thermal cycler (PEQLAB Ltd., Fareham, U.K.). PCR products were checked and purified using a PCR clean-up Gel Extraction NucleoSpin ® Gel and a PCR Clean-up Kit (Macherey-Nagel, Düren, Germany), according to the manufacturer's instructions. Following final purification, the PCR products were directly sequenced. Sequencing reactions were carried out and the above-mentioned PCR primers were employed to automatically determine the sequences in the Genetic Analyzer at the 1st Base Company (Kembangan, Malaysia).

Sequence Alignment and Phylogenetic Analyses
The resulting tef-1, cam, and rpb2 sequences were assessed for similarity analysis via the BLAST program available from the NCBI (http://blast.ncbi.nlm.nih.gov, accessed on 10 December 2022). Multiple sequence alignment was performed using MUSCLE [89], and any necessary modifications were made using BioEdit version 6.0.7. [90]. The combined data set of tef-1, cam, and rpb2 data was employed to conduct a multi-gene phylogenetic analysis. Phylogenetic trees were constructed using the maximum likelihood (ML) and Bayesian inference (BI) methods. The ML analysis was performed using 25 categories and 1000 bootstrap (BS) replicates with the GTRCAT model of nucleotide substitution [91] on RAxML-HPC2 version 8.2.12 [92] at the CIPRES web portal [93]. The optimal model for substitution of nucleotides was derived using the jModeltest v.2.3 [94] according to the Akaike Information Criterion (AIC) method. BI analysis was performed using the MrBayes v. 3.2.6 software [95]. For BI analysis, six simultaneous Markov chains with random starting trees were run for a million generations, with 1000 generations of each chain being sampled. The first 2000 trees were removed using a burn-in phase, and then the remaining trees were utilized to construct a phylogenetic tree using the 50% majority rule consensus. The Bayesian posterior probabilities (PPs) were subsequently calculated. The phylogenetic trees were visualized using FigTree v1.4.0 [96].

Conclusions
Watermelon fruit rot caused by Fusarium species is typically spread either in the field or during storage and is occurring in many countries around the world. In the present study, we reported F. compactum and F. paranaense to be pathogens of watermelon fruit rot for the first time, in Thailand and worldwide. These fungi were obtained from rot lesions taken from watermelons and identified on the basis of morphological features and multi-gene phylogenetic analyses. In pathogenicity tests under artificial inoculation conditions, the same symptoms as those seen during the postharvest storage period were observed. Therefore, F. compactum and F. paranaense were concluded to be novel pathogens of fruit rot diseases in watermelons. Further investigation of the epidemiology of these diseases in other areas of Thailand, as well as for the purposes of establishing effective management practices, is required. Moreover, in the future, the development of efficient monitoring and preventative strategies will be necessary in order to prevent the significant financial losses introduced by fruit rot disease.